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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION

International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

C07K 14^53, 14/555, 1/107, A61K 47/48

(11) International Publication Number:
(43) International Publication Date:

WO 9rV11953

25 April 1996 (25.04.96)

(21) International Application Number:

PC17US95/0I729

(22) International Filing Date:

8 February 1995 (08.02.95)

(30) Priority Data:

08/321 ,5 10 '

12 October 1994 (12.10.94)

(71) Applicant: AMGEN INC. [US/US]; Amgen Center, 1840

Dehavilland Drive, Thousand Oaks, CA 91320-1789 (US).

(72) Inventors: KINSTLER, Olaf, B.; Unit A, 533 North Oaktree,

Thousand Oaks, CA 91360 (US). GABRIEL, Nancy, E.;
3501 Bear Creek Court, Newbury Park, CA 91320 (US).
FARRAR, Christine. E.; 667 Valley Oak Lane, Newbury
Park, CA 91320 (US). DEPRINCE, Randolph, B.; 129
Hartland Court, Raleigh, NC 27614 (US).

(74) Agents: ODRE, Steven, M. et a].; Amgen Inc., Amgen Center.
1840 Dehavilland Drive. Thousand Oaks, CA 91320-1789
(US).

(81) Designated States: AM, AT, AU, BB, BG. BR, BY, CA, CH,
CN. CZ, DE, DK, EE, ES, FI. GB, GE. HU, JP, KE, KG,
KP, KR, KZ, LK, LR, LT. LU, LV, MD, MG, MN, MW,
MX. NL, NO, NZ, PL, PT, RO, RU, SD, SE, SI. SK, TJ,
TT, UA, UZ, VN, European patent (AT, BE. CH. DE, DK,
ES. FR. GB, GR, IE, IT, LU, MC, NL, PT, SE). OAPI
patent (BF, BJ, CF, CG. CI, CM, GA, GN, ML, MR, NE,
SN, TD, TG). ARIPO patent (KE, MW, SD, SZ).

Published

With international search report.

(54) Title: N-TERMINALLY CHEMICALLY MODIFIED PROTEIN COMPOSITIONS AND METHODS
(57) Abstract

Provided herein are methods and compositions relating to the attachment of water soluble polymers to proteins. Provided are novel
methods for N-terminally modifying proteins or analogs thereof, and resultant compositions, including novel N-terminally chemically
modified G-CSF compositions and related methods of preparation. Also provided is chemically modified consensus interferon.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Austria

linked Kingdom

Mauritania

Australia

Georgia

Malawi

Barbados

Guinea

Niger

Greece

Netherlands

Burkina Ftso

Hungary

Norway

Bulgaria

Ireland

New Zealand

Benin

Italy

Poland

Brazil

Japan

Portugal

Bclaras

Kenya

Romania

Canada

Kyrgystan

Russian Federation

Centra] African Republic

Democratic People' • Republic

Sudan

Congo

of Korea

Sweden

Switzerland

Republic of Korea

Slovenia

Cted'Ivoire

Kazakhstan

Slovakia

^^awmooo

Liechtenstein

Senegal

China

Sri Lanka

Chad

Czechoslovakia

Luxembourg

Togo

Czech Republic

Latvia

Tajikistan

Germany

Monaco

Trinidad and Tobago

Denmark

Republic of Moldova

Ukraine

Spam

Madagascar

United States of America

Finland

Mali

Uzbekistan

France

Mongolia

Viet Nam

Gabon

WO 96/11953

PCT/US95/01729

-1-

N-TERMINALLY CHEMICALLY MODIFIED PROTEIN
COMPOSITIONS AND METHODS

Field of the Invention

5 The present invention broadly relates to the

field of protein modification, and, more specifically,
the attachment of water soluble polymers to proteins or
analogs thereof (the term "protein" as used herein is
synonymous with "polypeptide" or "peptide" unless

10 otherwise indicated) . The present invention also
relates to novel methods for N-terminally modifying
proteins or analogs thereof, and resultant compositions.
In another aspect, the present invention relates to
novel N-terminally chemically modified G-CSF

15 compositions and related methods of preparation. The
present invention also relates to chemically modified
consensus interferon.

Background

20 Proteins for therapeutic use are currently

available in suitable forms in adequate quantities
largely as a result of the advances in recombinant DNA
technologies. The availability of recombinant proteins
has engendered advances in protein formulation and

25 chemical modification. One goal of such modification is
protein protection. Chemical attachment may effectively
block a proteolytic enzyme from physical contact with
the protein backbone itself, and thus prevent
degradation. Additional advantages include, under

30 certain circumstances, increasing the stability and
circulation time of the therapeutic protein and
decreasing immunogenicity . A review article describing
protein modification and fusion proteins is Francis,
Focus on Growth Factors 2: 4-10 (May 1992) (published by

35 Mediscript, Mountview Court, Friern Barnet Lane, London
N20, OLD, UK) .

WO 96/1 1953 PCI7US95/01729

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Polyethylene glycol ("PEG") is one such

■

chemical moiety which has been used in the preparation
of therapeutic protein products (the verb "pegylate"
meaning to attach at least one PEG molecule) . For
5 example Adagen, a pegylated formulation of adenosine
deaminase is approved for treating severe combined
immunodeficiency disease; pegylated superoxide dismutase
has been in clinical trials for treating head injury;
pegylated alpha interferon has been tested in phase I

10 clinical trials for treating hepatitis; pegylated

glucocerebrosidase and pegylated hemoglobin are reported
to have been in preclinical testing. The attachment of
polyethylene glycol has been shown to protect against
proteolysis, Sada, et al., j # Fermentation

15 Bioengineering 21: 137-139 (1991), and methods for

attachment of certain polyethylene glycol moieties are
available. £££ U.S. Patent No. 4,179,337, Davis et al.,
M Non- Immunogenic Polypeptides, " issued December 18,
1979; and U.S. Patent No. 4,002,531, Royer, "Modifying

20 enzymes with Polyethylene Glycol and Product Produced
Thereby,- issued January 11, 1977. For a review, s&S.
Abuchowski et al., in Enzymes as Drugs. (J.S.
Holcerberg and J. Roberts, eds. pp. 367-383 (1981)).

Other water soluble polymers have been used,

25 such as copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol,
polyvinyl pyrrolidone, poly-1, 3-dioxolane,
poly-1,3, 6-trioxane, ethylene/maleic anhydride
copolymer, polyaminoacids (either homopolymers or random

30 copolymers) .

For polyethylene glycol, a variety of means
have been used to attach the polyethylene glycol
molecules to the protein. Generally, polyethylene
glycol molecules are connected to the protein via a
35 reactive group found on the protein. Amino groups, such

WO 96/1 1953 PCT/US95/01729

- 3 -

as those on lysine residues or at the N-terminus, are
convenient for such attachment. For example, Royer
(U.S. Pat. No. 4,002,531, above) states that reductive
alkylation was used for attachment of polyethylene
5 glycol molecules to an enzyme. EP 0 539 167, published
April 28, 1993, Wright, "Peg Imidates and Protein
Derivates Thereof states that peptides and organic
compounds with free amino group (s) are modified with an
immediate derivative of PEG or related water-soluble
10 organic polymers. U.S. Patent No. 4, 904,584, Shaw,

issued February 27, 1990, relates to the modification of
the number of lysine residues in proteins for the
attachment of polyethylene glycol molecules via reactive
amine groups.

15 One specific therapeutic protein which has

been chemically modified is granulocyte colony
stimulating factor, "G-CSF." G-CSF induces the rapid
proliferation and release of neutrophilic granulocytes
to the blood stream, and thereby provides therapeutic

20 effect in fighting infection.

European patent publication EP 0 401 384,
published December 12, 1990, entitled, "Chemically
Modified Granulocyte Colony Stimulating Factor,"
describes materials and methods for preparing G-CSF to

25 which polyethylene glycol molecules are attached.

Modified G-CSF and analogs thereof are also
reported in EP 0 473 268, published March 4, 1992,
entitled "Continuous Release Pharmaceutical Compositions
Comprising a Polypeptide Covalently Conjugated To A

30 Water Soluble Polymer," stating the use of various G-CSF
and derivatives covalently conjugated to a water soluble
particle polymer, such as polyethylene glycol.

A modified polypeptide having human
granulocyte colony stimulating factor activity is

35 reported in EP 0 335 423 published October 4, 1989.

WO 96/1 1953 PCT/US95/01729

- 4 -

Another example is pegylated IL-6, EP 0 442
724, entitled, "Modified hIL-6," (s&e co-pending
U.S. S.N. 07/632,070) which discloses polyethylene glycol
molecules added to IL-6.
5 EP 0 154 316, published September 11, 1985

reports reacting a lymphokine with an aldehyde of
polyethylene glycol.

Many methods of attaching a polymer to a
protein involve using a moiety to act as a linking

10 group. Such moieties may, however, be antigenic. A
tresyl chloride method involving no linking group is
available, but this method may be difficult to use to
produce therapeutic products as the use of tresyl
chloride may produce toxic by-products. See Francis et

15 al., In: Stability of protein pharmaceuticals; in vivo
pathways of degradation and strategies for protein
stabilization (Eds. Ahern., T. and Manning, M.C.)
Plenum, New York, 1991) Also, Delgado et al., "Coupling
of PEG to Protein By Activation With Tresyl Chloride,

20 Applications In Immunoaffinity Cell Preparation", In:
Fisher et al., eds., Separations Using Aqueous Phase
Systems, Applications In Cell Biology and Biotechnology,
Plenum Press, N. Y.N. Y. , 1989 pp. 211-213.

Chamow et al., Biocon jugate Chem. £: 133-14 0

25 (1994) report the modification of CD 4 immunoadhesin with
monomethoxlypoly (ethylene glycol) aldehyde via reductive
alkylation. The authors report that 50% of the CD4-Ig
was MePEG-modified under conditions allowing the control
over the extent of pegylation. Id. at page 137. The

30 authors also report that the in vitro binding capability
of the modified CD4-Ig (to the protein gp 120) decreased
at a rate correlated to the extent of MePEGylation.
Ibid. See Rose et al., Biocon jugate Chemistry 2:

154-159 (1991) which reports the selective attachment of

WO 96/1 1953 PCT/US9S/01729

- 5 -

the linker group carbohydrazide to the C-terminal
carboxyl group of a protein substrate (insulin) .

None of the methods in the general state of
the art, or the art relating to particular proteins,
5 allow for selective attachment of a water soluble

polymer to the N-terminus of a protein such as G-CSF,
however. Rather, the currently existing methods provide
for non-selective attachment at any reactive group,
whether located within the protein, such as a lysine

10 side group, or at the N-terminus. This results in a
heterogenous population. For example, for pegylated
G-CSF molecules, some molecules have a different number
of polyethylene glycol moieties than others. As an
illustration, protein molecules with five lysine

15 residues reacted in the above methods may result in a
heterogenous mixture, some having six polyethylene
glycol moieties, some five, some four, some three, some
two, some one and some zero. And, among the molecules
with several, the polyethylene glycol moieties may not

20 be attached at the same location on different molecules.

This is disadvantageous when developing a
therapeutic pegylated protein product. In such
development, predictability of biological activity is
crucial. For example, it has been shown that in the

25 case of nonselective conjugation of superoxide dismutase
with polyethylene glycol, several fractions of the
modified enzyme were completely inactive (P.McGoff et
al. Chem. Pharm. Bull. .26:3079-3091 (1988)). One cannot

have such predictability if the therapeutic protein
30 differs in composition from lot to lot. Some of the

polyethylene glycol moieties may not be bound as stably
in some locations as others, and this may result in such
moieties becoming dissociated with the protein. Of
course, if such moieties are randomly attached and
35 therefore become randomly dissociated, the

WO 96/11953 PCIYUS95/01729

- 6 -

pharmacokinetics of the therapeutic protein cannot be
precisely predictable. From a consumer's point of view,
the circulation time may vary from lot to lot, and thus
dosing may be inaccurate. From a producer's point of
5 view, garnering regulatory approval for sale of the
therapeutic protein may have added complexities.
Additionally, none of the above methods provide for
selective N-terminal chemical modification without a
linking moiety (between the protein and the polymer) .

10 If a linking moiety is used, there may be disadvantages
due to possible antigenicity.

Thus, there exists a need for methods allowing
for selectively N-terminally chemically modified
proteins and analogs thereof, including G-CSF and

15 consensus interferon (two chemically modified proteins
exemplified below) . The present invention addresses
this need in a number of aspects.

Summary of the Invention

20 The present invention relates to substantially

homogenous preparations of N-terminally chemically
modified proteins, and methods therefor. Unexpectedly,
chemical modification at the N-terminus of G-CSF
demonstrated advantages in stability which are not seen

25 in other G-CSF species containing one chemical

modification at another location on the molecule. Also
unexpectedly, in the present process for making
N-terminally chemically modified G-CSF, it was found
that using reductive alkylation, one could provide

30 conditions for selectively modifying the N-terminus, and
this method is broadly applicable to other proteins (or
analogs thereof), as well as G-CSF. Also surprisingly,
using reductive alkylation, the end product — protein
with an amine linkage to the water soluble polymer —

35 was found to be far more stable than identical

WO 96/1 1953

PCT/US95/01729

polymer/protein conjugate having an amide linkage. One
other protein so modified (as described in a working
example below) is consensus inter feron. Thus, as
described below in more detail, the present invention
5 has a number of aspects relating to chemically modifying
proteins (or analogs thereof) as well as specific
modifications of specific proteins.

In one aspect, the present invention relates
to a substantially homogenous preparation of

10 N-terminally chemically modified G-CSF (or analog

thereof) and related methods. One working example below
demonstrates that N-terminally monopegylated G-CSF more
stable than other types of monopegylated G-CSF.
Additionally, since the N-terminus of the G-CSF molecule

15 is more available during reaction with polyethylene
glycol, a higher proportion of the N-termini are
pegylated, and therefore, this species provides
processing advantages.

The present invention also relates to a type

20 of reductive alkylation which selectively activates

ct-amino group of the N-terminal residue of a protein or

analog thereof, thereby providing for selective
attachment of a water soluble polymer moiety at the
N-terminus. This provides for a substantially

25 homogenous preparation of polymer/protein conjugate

molecules as well as (if polyethylene glycol is used) a
preparation of pegylated protein molecules having the
polyethylene glycol moiety directly coupled to the
protein moiety. This method is described below for

30 G-CSF and for consensus interferon, and these provide
for additional aspects of the present invention.

WO 96/11953

PCT/US95/01729

Brigf Dfisrrl pi- ion of t hg Drawings

FIGURE 1A is a reproduction of the
chromatogram of the peaks from ion exchange
chromatography of pegylated G-CSF.
5 FIGURE IB is an SDS-PAGE of various species of

mono-pegylated G-CSF.

FIGURE 2 is an SEC-HPLC profile of (Line A)
recombinant human methionyl G-CSF standard; (Line B)
SCM-PEG-GCSF reaction mix; (Line C) N-terminally
10 pegylated G-CSF; (Line D) iysine 35 monopegylated G-CSF;
(Line E) lysine 41 monopegylated G-CSF.

FIGURES 3A, 3B, and 3C are HPLC endoproteinase
SV8 peptide mapping tracings of (3A) N-terminally
pegylated G-CSF; (3B) lysine 35 monopegylated G-CSF;
15 (3C) lysine 41 monopegylated G-CSF.

FIGURE 4 is a bar graph illustrating a
comparison of in vitro bioactivity of monopegylated G-
CSF species compared to an unpegylated standard.

FIGURES 5A and 5B are graphs illustrating
20 results of In vivo bioactivity assays of monopegylated
G-CSF derivatives, with 5A illustrating the average
hamster white blood cell count after a single
subcutaneous injection of N-terminally pegylated G-CSF,
lysine 35 monopegylated G-CSF, or lysine 41
25 monopegylated G-CSF, and 5B illustrating the net average
white blood cell count area under the curve after a
single subcutaneous injection of the various
monopegylated G-CSF derivatives listed above.

FIGURES 6A, 6B, and 6C are SEC-HPLC profiles
30 for stability studies of N-terminally pegylated G-CSF or
lysine 35 monopegylated G-CSF. FIGURES 6A and 6B are the
profiles for stability studies conducted at pH 6.0 at
4°C for (6A) N-terminally monopegylated G-CSF or (6B)
lysine 35 monopegylated G-CSF. FIGURE 6C shows the
35 profiles for extended stability studies at pH 6.0 and

WO 96/11953

PCT/US95/01729

4°C for lysine 35 monopegylated G-CSF. Time ( n T n )
indicates days.

FIGURE 7 illustrates size exclusion HPLC
analysis of the reaction mixture in the process of
5 reductive alkylation of rh-G-CSF with

methoxypolyethylene glycol aldehyde (MW 6 kDa) .

FIGURE 8 illustrates size exclusion HPLC
analysis of the reaction mixture using
N-hydroxysuccinimidyl ester of MPEG, also at MW«6kDa.
10 FIGURE 9 illustrates the total white blood

cell response after a single subcutaneous dose to mono-N
terminal MPEG-GCSF conjugates prepared by reductive
alkylation of rh-G-CSF with MPEG aldehydes of different
molecular weights (6 kDa,12kDa and 20 kDa).

Detailed Description

The present invention relates to substantially
homogenous preparations of N-terminally chemically
modified proteins, and methods therefor.

20 In one aspect, the present invention relates

to N-terminally chemically modified G-CSF compositions
and methods therefor.

The present methods (for both N-terminally
modified G-CSF as well as the present reductive

25 alkylation methods) provide for a substantially

homogenous mixture of monopolymer/protein conjugate.
"Substantially homogenous" as used herein means that the
only polymer/protein conjugate molecules observed are
those having one polymer moiety. The preparation may

30 contain unreacted (i.e., lacking polymer moiety)
protein. As ascertained by peptide mapping and
N-terminal sequencing, one example below provides for a
preparation which is at least 90% monopolymer/protein
conjugate, and at most 10% unreacted protein.

35 Preferably, the N-terminally monopegylated material is

WO 96/1 1953 PCT/US95/01729

- 10 -

at least 95% of the preparation (as in the working
example below) and most preferably, the N-terminally
monopegylated material is 99% of the preparation or
more. The monopolymer/protein conjugate has biological
5 activity. The present "substantially homogenous"
N-terminally pegylated G-CSF preparations provided
herein are those which are homogenous enough to display
the advantages of a homogenous preparation, e.g., ease
in clinical application in predictability of lot to lot

10 pharmacokinetics .

One may choose to prepare a mixture of
polymer/protein conjugate molecules, and the advantage
provided herein is that one may select the proportion of
monopolymer/protein conjugate to include in the mixture.

15 Thus, if desired, one may prepare a mixture of various
protein with various numbers of polymer moieties
attached (i.e., di-, tri-, tetra-, etc.) and combine
with the monopolymer/protein conjugate material prepared
. using the present methods, and have a mixture with a

20 predetermined proportion of monopolymer/protein
conjugate .

Provided below is a working example using
G-CSF, which, as described above, is a therapeutic
protein used to treat hematopoietic disorders. In

25 general, G-CSF useful in the practice of this invention
may be a form isolated from mammalian organisms or,
alternatively, a product of chemical synthetic
procedures or of prokaryotic or eukaryotic host
expression of exogenous DNA sequences obtained by

30 genomic or cDNA cloning or by DNA synthesis. Suitable
prokaryotic hosts include various bacteria (e.g.,
fi. ; suitable eukaryotic hosts include yeast (e.g.,

S. cerevisiae l and mammalian cells (e.g., Chinese
hamster ovary cells, monkey cells) . Depending upon the

35 host employed, the G-CSF expression product may be

WO 96/11953

PCT/US95/01729

glycosylated with mammalian or other eukaryotic
carbohydrates, or it may be non-glycosylated. The G-CSF
expression product may also include an initial
methionine amino acid residue (at position -1) . The
5 present invention contemplates the use of any and all
such forms of G-CSF, although recombinant G-CSF,
especially JL. eoli derived, is preferred, for, among
other things, greatest commercial practicality.

Certain G-CSF analogs have been reported to be

10 biologically functional, and these may also be

chemically modified, by, for example, the addition of
one or more polyethylene glycol molecules. G-CSF
analogs are reported in U.S. Patent No. 4,810,643.
Examples of other G-CSF analogs which have been reported

15 to have biological activity are those set forth in

AU-A-76380/91, EP 0 459 630, EP 0 272 703, EP O 473 268
and EP O 335 423, although no representation is made
with regard to the activity of each analog reportedly
disclosed. Sfifi also AU-A-10948/92, PCT US94/00913 and EP

20 0 243 153.

Generally, the G-CSFs and analogs thereof

useful in the present invention may be ascertained by

practicing the chemical modification procedures as

provided herein to selectively chemically modify the
25 N-terminal a-amino group, and testing the resultant

product for the desired biological characteristic, such
as the biological activity assays provided herein. Of
course, if one so desires when treating non-human
mammals, one may use recombinant non-human G-CSF 1 s, such

30 as recombinant murine, bovine, canine, etc. See PCT WO
9105798 and PCT WO 8910932, for example.

Thus, another aspect of the present invention
includes N-terminally chemically modified G-CSF analog
compositions. As described above, G-CSF analogs may

35 include those having amino acid additions, deletions

WO 96/1 1953 PCT/US95/01729

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and/or substitutions (as compared to the G-CSF amino
acid sequence set forth in Example 1, below) . Those
G-CSF analogs which are predicted to function when
N-terminally pegylated to selectively stimulate the
5 production of neutrophils are those with an N-terminus
which is not necessary for binding to a G-CSF receptor.
Sfifi Hill et al., PNAS-USA 5167-5171 (1993); jsfifi also

PCT US94/00913.

The polymer molecules used may be selected

10 from among water soluble polymers, (For the reductive
alkylation procedure described herein, the polymers
should have a single reactive aldehyde,) The polymer
selected should be water soluble so that the protein to
which it is attached does not precipitate in an aqueous

15 environment, such as a physiological environment. For
reductive alkylation, the polymer selected should have a
single reactive aldehyde so that the degree of
polymerization may be controlled as provided for in the
present methods. The polymer may be branched or

20 unbranched. Preferably, for therapeutic use of the
end-product preparation, the polymer will be
pharmaceutical^ acceptable. One skilled in the art
will be able to select the desired polymer based on such
considerations as whether the polymer/protein conjugate

25 will be used therapeutically, and if so, the desired

dosage, circulation time, resistance to proteolysis, and
other considerations. For G-CSF, these may be
ascertained using the assays provided herein, and one
skilled in the art should select the appropriate assays

30 for other therapeutic proteins. The water soluble

polymer may be selected from the group consisting of,
for example, those listed above (in the Background
section), and dextran or poly(n-vinyl
pyrrolidone) polyethylene glycol, propropylene glycol

35 homopolymers, prolypropylene oxide/ethylene oxide

WO 96/11953

PCT/US95/01729

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co-polymers, polyoxyethylated polyols and polyvinyl
alcohol .

Subject to considerations for optimization as
discussed below, the polymer may be of any molecular
5 weight, and may be branched or unbranched. For

polyethylene glycol, the preferred molecular weight is
between about 2kDa and about lOOkDa (the term "about"
indicating that in preparations of polyethylene glycol,
some molecules will weigh more, some less, than the

10 stated molecular weight) . Examples 1 and 2 below

involve the use of PEG 6000, which was selected for ease
in purification and for providing an adequate model
system. Other sizes may be used, depending on the
desired therapeutic profile (e.g., the duration of

15 sustained release desired, the effects, if any on

biological activity, the ease in handling, the degree or
lack of antigenicity and other known effects of the
polyethylene glycol to a therapeutic protein or analog) .

One specific aspect of the present invention

20 is N-terminally monopegylated G-CSF comprised of a

polyethylene glycol moiety and a G-CSF moiety. For the
present compositions, one may select from a variety of
polyethylene glycol molecules (by molecular weight,
branching, etc.), the proportion of polyethylene glycol

25 molecules to G-CSF protein molecules in the reaction
mix, the type of pegylation reaction to be performed,
the method of obtaining the selected N-terminally
pegylated G-CSF, and the type of G-CSF to be used.
Further, the present compositions and methods include

30 formulation of pharmaceutical compositions, methods of
treatment and manufacture of medicaments.

The proportion of polyethylene glycol
molecules to protein molecules will vary, as will their
concentrations in the reaction mixture. In general, the

35 optimum ratio (in terms of efficiency of reaction in

WO 96/11953

PCT/US95/01729

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that there is no excess unreacted protein or polymer)
will be determined by the molecular weight of the
polyethylene glycol selected. In addition, as one
example of the present methods involves non-specific
5 pegylation and later purification of N-terminally
monopegylated species, the ratio may depend on the
number of available reactive groups (typically «or 3

amino groups) available. One working example herein
involved a fairly low reaction ratio of protein: PEG

10 molecules to obtain monopegylated material generally
(1.5 PEG molecules per protein molecules) .

For obtaining N-terminally pegylated G-CSF,
the method for pegylation may also be selected from
among various methods, as discussed above, or the

15 present reductive alkylation as described in Example 2,
below. A method involving no linking group between the
polyethylene glycol moiety and the protein moiety is
described in Francis et al., In: Stability of protein
pharmaceuticals: in vivo pathways of degradation and

20 strategies for protein stabilization (Eds. Ahern., T.
and Manning, M.C.) Plenum, New York, 1991) Also,
Delgado et al., "Coupling of PEG to Protein By
Activation With Tresyl Chloride, Applications In
Immunoaffinity Cell Preparation", In: Fisher et al.,

25 eds., Separations Using Aqueous Phase Systems,

Applications In Cell Biology and Biotechnology, Plenum
Press, N.Y.N.Y.,1989 pp. 211-213, involves the use of
tresyl chloride, which results- in no linkage group
between the polyethylene glycol moiety and the protein

30 moiety. This method may be difficult to use to produce
therapeutic products as the use of tresyl chloride may
produce toxic by-products. One of the present working
examples involves the use of N-hydroxy succinimidyl
esters of carboxymethyl methoxy polyethylene glycol. As

35 will be discussed in more detail below, another working

WO 96/11953 FCTAJS9S/01729

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example involves the use of the present reductive
alkylation methods.

The method of obtaining the N-terminally
pegylated G-CSF preparation (i.e., separating this
5 moiety from other monopegylated moieties if necessary)
may be by purification of the N-terminally pegylated
material from a population of pegylated G-CSF molecules.
For example , presented below is an example where
pegylated G-CSF is first separated by ion exchange

10 chromatography to obtain material having a charge
characteristic of monopegylated material (other
multi-pegylated material having the same apparent charge
may be present) , and then the monopegylated materials
are separated using size exclusion chromatography. In

15 this way f N-terminally monopegylated G-CSF was separated
from other monopegylated species, as well as other
multi-pegylated species. Other methods are reported.
For example, PCT WO 90/04606, published May 3, 1990,
reports a process for fractionating a mixture of PEG-

20 protein adducts comprising partitioning the PEG/protein
adducts in a PEG-containing aqueous biphasic system.

In a different aspect, the present invention
provides a method for selectively obtaining an
N-terminally chemically modified protein (or analog) .

25 Provided below is a method of protein modification by
reductive alkylation which exploits differential
reactivity of different types of primary amino groups
(lysine versus the N-terminal) available for
derivatization in a particular protein. Under the

30 appropriate reaction conditions, substantially selective
derivatization of the protein at the N- terminus with a
carbonyl group containing polymer is achieved. The
reaction is performed at pH which allows one to take
advantage of the pK a differences between the e-amino

35 groups of the lysine residues and that of the a-amino

WO 96/11953 PCT/US95/01729

- 16 -

group of the N-terminal residue of the protein. By such
selective derivatization attachment of a water soluble
polymer to a protein is controlled: the conjugation
with the polymer takes place predominantly at the
5 N-terminus of the protein and no significant

modification of other reactive groups, such as the
lysine side chain amino groups, occurs.

Importantly, and surprisingly, the present
invention provides for a method of making a

10 substantially homogenous preparation of

monopolymer /protein conjugate molecules, in the absence
of further extensive purification as is required using
other chemical modification chemistries. Additionally,
the product having an amine linkage is unexpectedly more

15 stable than a product produced with an amide linkage,
and this is demonstrated in the aggregation studies
below. More specifically, if polyethylene glycol is
used, the present invention also provides for
N-terminally pegylated protein lacking possibly

20 antigenic linkage groups, and having the polyethylene
glycol moiety directly coupled to the protein moiety
without toxic by-products.

The reaction may be diagrammed as follows
(indicating sodium cyanohydroboride as an illustrative.

25 reducing agent) :

WO 96/11953 PCT/US95/01729

- 17 -

WO 96/11953 PCT/US95/01729

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Thus, one aspect of the present invention is a

method for preparing a polymer/protein conjugate

comprised of (a) reacting a protein moiety having more

than one amino group with a water soluble polymer moiety

5 under reducing alkylation conditions, at a pH suitable
to selectively activate the a -amino group at the amino

terminus of said protein moiety so that said water
soluble polymer selectively attaches to said a -amino

group; and (b) obtaining the reaction product. One may

10 optionally, and preferably for a therapeutic product,

separate the reaction products from unreacted moieties.

Another aspect of the present invention is

that such reductive alkylation will provide for

selective attachment of the polymer to any protein
15 having an a -amino group at the amino terminus, and

provide for a substantially homogenous preparation of
monopolymer/ protein conjugate. The term "monopolymer/
protein conjugate" is used here to mean a composition
comprised of a single polymer moiety attached to a

20 protein moiety (also encompassed are those conjugates
using protein analogs as described herein) . The
monopolymer /protein conjugate will have a polymer moiety
located at the N-terminus, but not on amino side groups,
such as those for lysine. The preparation will

25 preferably be greater than 80% monopolymer/ protein
conjugate, and more preferably greater than 95%
monopolymer protein conjugate.

For a substantially homogenous population of
monopolymer /protein conjugate molecules, the reaction

30 conditions are those which permit the selective

attachment of the water soluble polymer moiety to the
N-terminus of the desired protein. Such reaction
conditions generally provide for pK a differences between
the lysine amino groups and the a-amino group at the

35 N-terminus (the pK being the pH at which 50% of the

WO 96/11953

PCT/US95/017Z9

amino groups are protonated and 50% are not) . In
general, for different proteins, different pH's may be
used for optimally modifying the a-amino groups of the

N-terminus.

5 The pH also affects the ratio of polymer to

protein to be used. In general, if the pH is lower than
the pK, a larger excess of polymer to protein will be
desired (i.e., the less reactive the N-terminal a-amino

group, the more polymer needed to achieve optimal

10 conditions) . If the pH is higher than the pK, the

polymer :protein ratio need not be as large (i.e., more
reactive groups are available, so fewer polymer
molecules are needed) .

Another important consideration is the

15 molecular weight of the polymer. In general, the higher
the molecular weight of the polymer, the fewer number of
polymer molecules which may be attached to the protein.
Similarly, branching of the polymer should be taken into
account when optimizing these parameters. Generally,

20 the higher the molecular weight (or the more branches)
the higher the polymer rprotein ratio.

For the present reductive alkylation, the
reducing agent should be stable in aqueous solution and
preferably be able to reduce only the Schif f base formed

25 in the initial process of reductive alkylation.

Preferred reducing agents may be selected from the group
consisting of sodium borohydride, sodium
cyanoborohydride, dimethylamine borate, timethylamine
borate and pyridine borate. Sodium cyanoborohydride was

30 used in the working examples below.

The water soluble polymer may be of the type
described above, and should have a single reactive
aldehyde for coupling to the protein. For polyethylene
glycol, use of PEG 6000 for coupling to G-CSF and PEG

35 12000 for consensus interferon are described below. It

WO 96/1 1953 PCT/US95/01729

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is noted, that for G-CSF, PEG 12000, 20000 and 25000
have also been used successfully in the present methods.
Polyethylene glycol propionaldenhyde ( see r e.g., U.S.

Patent No. 5,252,714) is advantageous for its stability
5 in water.

As indicated above, the present methods are

broadly applicable to any protein or analog thereof
having an N-terminal a-amino group. For example,

proteins which are the product of an exogenous DNA
10 sequence expressed in bacteria may have, as a result of
bacterially expression, an N-terminal methionyl residue
with an a-amino group. As indicated above, peptides are

included, as are peptidomimetics and other modified
proteins. Protein analogs, such as the G-CSF analogs
15 described above, and the non-naturally occurring

consensus interferon are also suitable for the present
methods .

Thus, for the present N-terminally chemically
modified G-CSF, any of the G-CSF 1 s or analogs as

20 described herein may be used (e.g., those described
.Slffira) . The working examples below use recombinant
G-CSF produced in bacteria, having 174 amino acids and
an extra N-terminal methionyl residue. As described
herein, the chemical modification may be performed with

25 any of the water soluble polymers described herein, and
the present working examples describe the use of
polyethylene glycol.

Consensus interferon is another protein used
in the present working examples. Demonstrated below is

30 the preparation of chemically modified consensus
interferon using the present reductive alkylation
methods for N-terminal monopegylation. Thus, other
aspects of the present invention relate to these
preparations. As employed herein, consensus human

35 leukocyte interferon, referred to here as "consensus

WO 96/11953

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interferon," or "IFN-con", means a nonnaturally-
occurring polypeptide, which predominantly includes
those amino acid residues that are common to all
naturally-occurring human leukocyte interferon subtype
5 sequences and which include, at one or more of those
positions where there is no amino acid common to all
subtypes, an amino acid which predominantly occurs at
that position and in no event includes any amino acid
residue which is not extant in that position in at least
10 one naturally-occurring subtype- IFN-con encompasses
the amino acid sequences designated IFN-coni, IFN-con2
and IFN-con3 which are disclosed in commonly owned U.S.
Patents 4,695,623 and 4,897,471, the entirety of which

are hereby incorporated by reference. (U.S. Patent Nos.
15 4,897,471 and 4,695,623 use the denomination "a" which

is not used herein.) DNA sequences encoding IFN-con may
be synthesized as described in the above-mentioned
patents or other standard methods. IFN-con polypeptides
are preferably the products of expression of

20 manufactured DNA sequences, transformed or transfected
into bacterial hosts, especially £. That is,

IFN-con is recombinant IFN-con. IFN-con is preferably
produced in £L. coll may be purified by procedures known
to those skilled in the art and generally described in

25 Klein et al.,J. Chromatog. 4M: 205-215 (1988) for

IFN-coni. Purified IFN-con may comprise a mixture of
isoforms, e.g., purified IFN-coni comprises a mixture of
methionyl IFN-coni, des-methionyl IFN-coni and
des-methionyl IFN-coni with a blocked N-terminus (Klein

30 et al., Arc. Biochem. Biophys. 22£: 531-537 (1990)).

Alternatively, IFN-con may comprise a specific, isolated
isoform. Isoforms of IFN-con are separated from each
other by techniques such as isoelectric focusing which
are known to those skilled in the art.

WO 96/1 1953 PCIYUS95/01729

- 22 -

Thus, another aspect of the present invention
is a chemically modified consensus interferon wherein
said consensus interferon moiety is selected from the
group consisting of IFN-coni, IFN-con2, and IFN-con3.

5 The chemical modification is using a water soluble

polymer as described herein, such as PEG, and the

present reductive alkylation methods may be used for

selective N-terminal chemical modification. Example 3
herein illustrates a chemically modified I FN coni

10 comprised of an I FN coni moiety connected at the

N-terminus to a polyethylene glycol moiety (PEG 12000) .

In another aspect, the present methods yield
pegylated proteins where the polyethylene glycol moiety
is directly attached to a protein moiety, and a separate

15 linking group is absent and no toxic by-products are
present. The examples include G-CSF and consensus
interferon as described herein. For a population of
pegylated G-CSF protein molecules wherein the
polyethylene glycol moiety is directly attached to the

20 G-CSF protein moiety (not necessarily a population of

N-terminally pegylated G-CSF molecules) , one may perform
the above reductive alkylation with or without an acidic
pH.

In yet another aspect of the present
25 invention, provided are pharmaceutical compositions of
the above. Such pharmaceutical compositions may be for
administration for injection, or for oral, pulmonary,
nasal or other forms of administration. In general,
comprehended by the invention are pharmaceutical
30 compositions comprising effective amounts of

monopolymer /protein conjugate products of the invention
together with pharmaceutically acceptable diluents,
preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers. Such compositions include diluents of
35 various buffer content (e.g., Tris-HCl, acetate,

WO 96/11953

PCT/US95/01729

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phosphate) , pH and ionic strength; additives such as
detergents and solubilizing agents (e.g., Tween 80,
Polysorbate 80), anti-oxidants (e.g., ascorbic acid,
sodium metabisulfite) , preservatives (e.g., Thimersol,
5 benzyl alcohol) and bulking substances (e.g., lactose,
mannitol); incorporation of the material into
particulate preparations of polymeric compounds such as
polylactic acid, polyglycolic acid, etc. or into
liposomes. Such compositions may influence the physical

10 state, stability, rate of in vivo release, and rate of
in vivo clearance of the present N-terminally chemically
modified proteins. See , fi^., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing
Co., Easton, PA 18042) pages 1435-1712 which are herein

15 incorporated by reference.

In yet another aspect of the present
invention, methods of treatment and manufacture of a
medicament are provided. Conditions alleviated or
modulated by the administration of the present

20 polymer/G-CSF conjugates (or analogs having the
hematopoietic biological properties of naturally
occurring G-CSF) are typically those characterized by a
reduced hematopoietic or immune function, and, more
specifically, a reduced neutrophil count. Such

25 conditions may be induced as a course of therapy for
other purposes, such as chemotherapy or radiation
therapy. Such conditions may result from infectious
disease, such as bacterial, viral, fungal or other
infectious disease. For example, sepsis results from

30 bacterial infection. Or, such condition may be

hereditary or environmentally caused, such as severe
chronic neutropenia or leukemias. Age may also play a
factor, as in the geriatric setting, patients may have a
reduced neutrophil count or reduced neutrophil

35 mobilization. Some of such conditions are reviewed in

WO 96/11953 PCIYUS95/01729

- 24 -

Filgrastim (r-met Hu G-CSF) in Clinical Practice,
Morstyn, G. and T.M. Dexter, eds., Marcel Dekker, Inc.,
N.Y., N.Y. (1993), 351 pp. Other less-studied
conditions which may be alleviated or modulated by
5 administration of the present polymer/G-CSF conjugates
may include the reduction of lipids (or cholesterol) in
the blood stream, and certain cardiovascular conditions,
as G-CSF may induce production of plasminogen
activators. The mode of action of G-CSF (or analogs) in

10 these settings is not well understood at present. The
addition of a water soluble polymer, such as
polyethylene glycol, may provide practical patient
benefits in that the sustained duration of biological
activity may allow for fewer G-CSF injections per course

15 of treatment.

Generally, conditions which may be alleviated
or modulated by administration of the present
polymer/consensus interferon are those to which
consensus interferon is applicable and include cell

20 proliferation disorders, viral infections, and
autoimmune disorders such as multiple sclerosis.
Cf . , McManus Balmer, DICP, The Annals of Pharmacotherapy
24 : 761-767 (1990) (Clinical use of biologic response
modifiers in cancer treatment: an overview. Part I. The

25 Interferons). Methods and compositions for the
treatment of cell proliferation disorders using
consensus interferon are described in PCT WO 92/06707,
published April 30, 1992, which is herein incorporated
by reference. For example, hepatitis (A, B, C, D, E)

30 may be treatable using the present pegylated consensus
interferon molecules. The working example below
demonstrates that, in vitro, chemically modified
consensus interferon has 20% of the biological activity
of non-chemically modified consensus interferon..

WO 96/11953

PCT/US95/01729

- 25 -

■

For all of the above molecules, as further
studies are conducted, information will emerge regarding
appropriate dosage levels for treatment of various
conditions in various patients, and the ordinary skilled
5 worker, considering the therapeutic context, age and
general health of the recipient, will be able to
ascertain proper dosing. Generally, for injection or
infusion, dosage will be between 0.01 Mg/kg body weight,
(calculating the mass of the protein alone, without
10 chemical modification) , and 100 Hg/kg (based on the
same) .

The below examples illustrate the various
aspects discussed above* In Example 1, the advantages of
N-terminally pegylated G-CSF are demonstrated as

15 compared to G-CSF monopegylated at lysine-35 or lysine
41 (of the G-CSF met + 174 amino acid version). Example
2 illustrates the present reductive alkylation in
N-terrainally pegylating G-CSF. The method provides for a
substantially homogenous preparation of N-terminally

20 pegylated G-CSF. Example 3 illustrates the present
reductive alkylation in N-terminally pegylating
consensus interferon .

EXAMPLE 1

A. Preparation of Recombinant Human met-G-CSF
Recombinant human met-G-CSF (referred to as
w rhG-CSF M or "r-met-hu-G-CSF" from time to time herein)
was prepared as described above according to methods in
30 the Souza patent, U.S. Pat. No., 4,810,643, which is

herein incorporated by reference. The rhG-CSF employed
was an EL- Cflli derived recombinant expression product
having the amino acid sequence (encoded by the DNA

WO 96/1 1953 PCT/US95/01729

- 26 -

sequence) shown below (Seq.ID NOs. 1 and 2):

ATG

ACT

CCA

TTA

GGT

CCT

GCT

TCT

CTG

CCG

CAA

AGC

TTT

CTG

AAA

TGT

CTG

GAA

^m^m mm m

CAG

GTT

CGT

AAA

• mm mm m

ATC

CAG

GGT

^m^m m>

GAC

GGT

GCT

GCA

CTG

CAA

GAA

AAA

f wh m

CTG

TGC

GCT

WX^ A

ACT

TAC

AAA

CTG

TGC

PAT

CPG

WWW

GAA

***** mm •

GAG

^mnm^0

CTG

GTA

\mf m\ mm

CTG

w A W

CTG

GGT

CAT

wA *

TPT

PTT

w ■* A

ATP

PPf5

www

1 w

ww 1

mm %r

CCG

CTG

TCT

m» A

TCT

TGT

CCA

w w*»

TCT

X w 1

CAA

GCT

PTT

w A A

PAR

PTfi
w 1 w

rpt

ww A

ww J>

CTG

TCT

CAA

CTG

CAT

TCT

GGT

CTG

TTC

CTG

TAT

CAG

GGT

CTT

CTG

CAA

GCT

CTG

GAA

GGT

ATC

TCT

CCG

GAA

CTG

GGT

CCG

ACT

CTG

GAC

ACT

CTG

CAG

CTA

GAT

GTA

GCT

GAC

TTT

GCT

ACT

ATT

TGG

CAA

CAG

ATG

GAA

GAG

CTC

GGT

ATG

GCA

CCA

GCT

CTG

CAA

CCG

ACT

CAA

GGT

GCT

ATG

CCG

GCA

TTC

GCT

TCT

GCA

TTC

CAG

CGT

GCA

GGA

GGT

GTA

CTG

GTT

GCT

TCT

CAT

CTG

CAA

TCT

TTC

CTG

GAA

GTA

TCT

TAC

CGT

GTT

CTG

CGT

CAT

CTG

GCT

CAG

CCG

TAA

TAG

(This was also the non-pegylated composition used for
40 the control animals.) Alternatively one may use
purchased Neupogen® for the following pegylation
procedures (the package insert • for which is herein
incorporated by reference) .

45 B. Preparation of Peavlatfiri r,-P:SP

A 10 mg/ml solution of the above rh-G-CSF, in
100 mM Bicine pH 8.0, was added to solid SCM-MPEG
(N-hydroxy succinimidyl esters of carboxymethyl methoxy
polyethylene glycol) (Union Carbide) with an average

WO 96/11953 PCT/US95/01729

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molecular weight of 6000 Daltons. This gave a 1.5 molar
excess of SCM-MPEG to rh-G-CSF. After one hour with
gentle stirring, the mixture was diluted to 2 mg/ml with
sterile water, and the pH was adjusted to 4.0 with
5 dilute HC1. The reaction was carried out at room
temperature. At this stage, the reaction mixture
consisted mainly of three forms of mono-pegylated rh-G-
CSF, some di-pegylated rh-G-CSF, unmodified rh-G-CSF and
reaction bi-product (N-hydroxy succinimide) .

C. Preparation of Kf-terminallv Peavlated rh-G-CSF
The three forms of monopegylated rh-G-CSF were
separated from each other using ion exchange
chromatography. The reaction mixture was loaded (1 mg

15 protein/ml resin) onto a Pharmacia S Sepharose FF column
(Pharmacia XK50/30 reservoir, bed volume of 440 ml)
equilibrated in buffer A (20 mM sodium acetate, pH 4.0) .
The column was washed with 3 column volumes of buffer A.
The protein was eluted using a linear gradient from 0-

20 23% buffer B (20 mM sodium acetate, pH 4.0, 1M NaCl) in
15 column volumes. The column was then washed with one
column volume of 100% buffer B and reequilibrated with
3 column volumes of buffer A. The flow rate for the
entire run was maintained at 8 ml/min. The eluent was

25 monitored at 280 nm and 5 ml fractions were collected.
Fractions containing the individual monopegylated
species were pooled according to FIGURE 1A. These pools
were concentrated with a 350 mL Amicon stirred cell
using a YM10 76 mm membrane.

30 Pooled fractions from the ion exchange

chromatography were subjected to size exclusion
chromatography to separate di-pegylated species from
monopegylated species. Typically, 5-10 mg in 2-5 ml of
solution were loaded onto a 120 ml Pharmacia Superdex

35 75 HR 16/60 column equilibrated with 20 mM sodium

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acetate pH 4.0. The column was run at 1.5 ml/min for
100 min. Two ml fractions were collected. The protein
content of the eluent was monitored at 280 nnw
Fractions from separated peaks were pooled and subjected
5 to analysis. The table below compares the proportional
yields for each peak.

TABLE 1

RAiaiHvP Yields and Site of Modification

Site of Modification

Figure 1A
Reference

Relative Yields

N-Terminus

Peak 1A

Lvsine-35

Peak 2A

Lvsine-41

Peak 3A

Under these conditions, the lysines at
positions 17 and 24 probably were not significantly
pegylated.

15 D. Characterization

Five analyses were done to characterize each
sample: (1) SDS-Page (Figure IB) , (2) Size exclusion
chromatography HPLC ("SEC HPLC") (Figure 2) , (3) peptide
mapping analysis (Figures 3A f 3B f and 3C),(4) In yltXQ

20 G-CSF bioassay (Figure 4), and (5) in yjvo testing in

hamster (Figures 5A and 5B) .

With regard to the composition of each sample,
results demonstrate that r of the N-terminally
monopegylated G-CSF, the samples showed a greater than

25 95% N-terminally pegylated composition, with the

remainder probably being unpegylated material (although
the remainder of the samples is lower than the detection
limit of the assay) . With regard to the percent
monopegylated for each of the three types of

30 monopegylated material <N-terminal, pegylated at lysine
35, and pegylated at lysine 41), the N-terminal and the
lysine 41 demonstrated greater than 97% monopegylated,

WO 9&1 1953

PCT/US9S/01729

- 29 -

and the lysine 35 pegylated material being somewhat
lower, probably due to the instability of the molecule
in the assay conditions. To summarize, the following
results were obtained:

TABLE 2

Percent Composition of

H-terminally pegylated G-CSF

Non-Reduced
SDS PAGE

SEC HPLC

N-Terminal
Sequencing*

Mono-pegylated
G-CSF

97.44

99.43

96.6

Unmodified
G-CSF

2.56

0.57

3.4

10 * The N-terminal sequencing, as discussed in£ta is not
here considered quantitative, as there may have been
artifactual separation of the polyethylene glycol
molecule from the N-terminus of the protein during the
sequencing process.

TABLE 3

Percent Monopegylated for Three Species

N-terminal

LYS35 PEG-

LYS41

PEG-GCSF

GCSF**

PEG-GCSF

(RI/UV=.96)*

(RI/UV=.72)

(RI/UV=1.12)

Non- reduced

•

SDS-PAGE

97.44

77.41

100.00

SEC HPLC

99.43

93.38

99.96

* RI/UV refers to the Index of Refraction/Ultraviolet

20 light absorbance ratio, and is used to estimate the

number of polyethylene glycol molecules per molecule of
protein. It is calculated from the SEC HPLC data using

WO 96/1 1953 PCT/US95/01729

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an Index of Refraction for polyethylene glycol and an
ultraviolet absorbance for protein.

** Note that this species is unstable under the assay
conditions used.

METHODS

1. SDS-PAGE . SDS-PAGE was carried out in a
non-reduced 4-20% ISS Daiichi Pure Chemicals, Co.,

10 Tokyo, Japan minigel using a Coomassie Brillant Blue
R-250 stain. The gel was scanned using a molecular
Dynamics Densitometer with Image Quant.
Results: Results are presented in FIGURE IB. Lane
number 1 (from the left hand side) included molecular

15 weight protein standards (Novex Mark 12 Molecular Weight
Standards) . Lane 2 contains 3 \ig rh-G-CSF standard.
Lane 3 contains the SCM-PEG-GCSF reaction mix, with 10
\lg loaded. Lane 4 contains N-terminally monopegylated
G-CSF, with 10 \ig loaded. Lane 5 contains 10 \ig of

20 monopegylated G-CSF with the pegylation site at the
lysine found at the 35th residue from the N-terminal
methionine. Lane 6 contains 10 \ig of monopegylated
G-CSF with the pegylation site at the lysine found at
the 41st residue from the N-terminal methionine. As can

25 be seen, Lane 3, containing the N-terminally

monopegylated material, shows a single band

2. Size Exclusion Chromatographv-High

Pressure Liquid Chromatography.. SEC-HPLC was carried

out using a Waters HPLC system with a Biosep SEC 3000
30 column, using 100 mM sodium phosphate, pH 6.9, Iml/min
for 20 minutes. The signal was monitored at 280 nm.
Results: As can be seen from Figure 2, line "C, w
containing the N-terminally monopegylated rh-G-CSF
contains a single peak, as do lines "D* (Lys-35
35 monopegylated material) and n E" (Lys-41 monopegylated

WO 96/11953

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material). This indicates substantial purity among the
separated fractions of monopegylated G-CSF.

3. Peptide mapping . The following methods

were used. Three samples, called "Mono -PEG 1", "Mono-
5 PEG-2", and "Mono-PEG-3", were analyzed, (a) Reductive
alkylation. 500 \ig aliquots of mono-PEG G-CSF were
speed vac dried and reconstituted to a concentration of
1 mg in 950 (ll in 0.3 M Tris-HCl containing 6 M
Guanidinum HC1 and 1 mM EDTA pH 8.4. Samples were then

10 S-carboxyraethylated by adding iodoacetic acid and

incubated at 37°C for 20 minutes. Samples were then
desalted using Sephadex G-25 Quick Spin Protein Columns
and buffer exchanged. After desalting and buffer
exchange, sample concentration was adjusted to 0.5 mg/ml

15 using additional buffer. (b) Endoproteinase SV8

digestion. Samples were digested with SV8 (enzyme to
substrate ratio of 1:25) at 25°C for 26 hours, (c) HPLC
peptide mapping. Protein digests were injected onto a
Vydac C4 column (4.6 x 250 mm, 5 \i particle size, 300 A

20 pore size) and peptides were mapped by HPLC using a

linear gradient of acetonitrile in 0.1% TFA. Peptides
were manually collected and dried in a Speed Vac for
sequence analysis. Results: As compared to a reference
standard, (i) (FIGURE 3A) for "Mono-PEG- 1", (the N-

25 terminally mono-pegylated material), a peak at 57.3
minutes diminished and a new peak appeared at 77.5
minutes; (ii) (FIGURE 3B) for "Mono-PEG-2", (the lysine
35 pegylated material) , there was a decrease in peak
height for a peptide with a retention time of 30.3

30 minutes, and a new peak eluted at 66.3 minutes; (iii)
(FIGURE 3C) for "Mono-PEG-3" (the lysine 41 pegylated
material), the peak at retention time of 30.3 minutes
was missing, and a new peak appeared at 66.4 minutes.
These peptides were the only significant differences in

35 the sample maps. There were some small incomplete

WO 96/1 1953

PCT/DS95/01729

cleavages seen on either side of the peptide at 86.1
minutes due to minor digestion differences, (d) N-
terminal sequence analysis. Each of the "new" peptides
in the above maps were N-terminally sequenced for
5 identification. The dried peptides were reconstituted
in 0.1% TFA and sequenced on an ABI protein sequencer.
For "Mono-PEG-l" (the N-terminally pegylated material),
60% of the -new- peak (at 77.5 minutes) was sequenced
for 10 cycles. The initial yield was less than 5%,

10 indicating that the N-terminal methionyl residue is

blocked by a polyethylene glycol molecule. It is noted
that this initial peptide should have resulted in a zero
initial yield, and the <5% yield observed may be from
detachment of the polyethylene glycol from the N-

15 terminal methionyl during sequence analysis. The

sequence detected was that of the N-terminal peptide,
M-T-P-L-G-P-A-S-S . For "Mono-PEG-2", (the lysine 35
pegylated material), 80% of the total peak volume was
collected for the peak at 66.3 minutes, and was

20 sequenced for 9 cycles. The recovery of lysine 35 was
significantly low, indicating pegylation at position 35.
The recovery of lysine 41 was consistent with the other
residue, indicating no modification of this position.
The peptide at 30.3 minutes decreased in peak height

25 compared to the corresponding peak in the standard
reference map. The peptide at 30.3 minutes is only
57.5% of the peak area of the corresponding peptide.
The sequence detected for this species was
K-L-C-A-T-Y-K-L. For "Mono-PEG-3", the lysine 41

30 material, 80% of the total peak volume collected for the
peptide eluting» at 66.4 minutes was sequenced for 9
cycles. The sequence detected was K-L-C-A-T* Y-K-L, and
contained lysine residues 35 and 41. The recovery of
lysine 35 was consistent with other residue recoveries.

35 The recovery of lysine 41 was significantly lower

WO 96/11953

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- 33 -

indicating pegylation at position 41. Results : "Mono-
PEG-1 W is N-terminal ly monopegylated material; "Mono-
PEG-2" is lysine 35 partially pegylated; and "Mono-PEG-
3" is lysine 41 pegylated material. By comparing both
5 the reference standard (non-pegylated G-CSF) and GCSF
monopegylated 1, 2, and 3 peptide maps, it was found
that both the "Mono-PEG-^" (lysine 35) and w Mono-PEG-3 w
(lysine 41) maps exhibit slightly diminished peak
heights for the N-terminal peptide- This indicates that

10 the lysine 35 and lysine 41 material contains a small
amount of N-terminally pegylated material or that the
N-terminal methionine has a small percentage of
pegylation.

4. In vitro activity. The material was

15 active. FIGURE 4 illustrates the results of in vitro
assays. As can be seen, the N-terminally monopegylated
material had 68% of the activity of non-modified
rhG-CSF.

Methods: The G-CSF in vitro bioassay is a mitogenic
20 assay utilizing a G-CSF dependent clone of murine 32D
cells. Cells were maintained in Iscoves medium
containing 5% FBS and 20 ng/ml rhG-CSF. Prior to sample
addition, cells were prepared by rinsing twice with
growth medium lacking rhG-CSF. An extended twelve point
25 rhG-CSF standard curve was prepared, ranging from 48 to
.5ng/ml (equivalent to 4800 to 50 IU/ml) . Four
dilutions, estimated to fall within the linear portion
of the standard curve, (1000 to 3000 IU/ml), were
prepared for each sample and run in triplicate. Because
30 of their apparent lower activity in vitro P the pegylated

rhG-CSF samples were diluted approximately 4-10 times
less. A volume of 40}il of each dilution of sample or

standard is added to appropriate wells of a 96 well

microtiter plate containing 10,000 cells/well. After
35 forty-eight hours at 37°C and 5.5% CO2, O.SjimCi of

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methyl- 3 H-thymidine was added to each well. Eighteen
hours later , the plates were then harvested and counted.
A dose response curve (log rhG-CSF concentration vs.
CPM-background) was generated and linear regression
5 analysis of points which fall in the linear portion of
the standard curve was performed. Concentrations of
unknown test samples were determined using the resulting
linear equation and correction for the dilution factor.
Results: Results are presented in FIGURE 4. As can be
10 seen, of the three monopegylated species, N-terminally
monopegylated G-CSF demonstrates the highest in vitro

biological activity.

5. Tn vivo activity. In vivo testing

confirmed the activity of the N-terminally pegylated

15 material. The in vivo testing was carried out by dosing
male golden hamsters with a 0.1 mg/kg of sample, using a
single subcutaneous injection. Four animals were
subjected to terminal bleeds per group per time point.
Serum samples were subject to a complete blood count on

20 the same day that the samples were collected. The

average white blood cell counts were calculated. As can
be seen in FIGURES 5A and 5B, the response from each
material peaks after one day following a single
subcutaneous injection of 0.1 mg/kg. Two of the

25 monopegylated materials, (N-terminal and Lys-35) showed
prolonged responses, while the response for the protein
pegylated at lysine-41 showed no increase in in vivo
activity over unmodified rhG-CSF (indeed it shows less,
FIGURE 5B) . These results illustrate that attaching a

30 single polyethylene glycol molecule can dramatically

alter the therapeutic profile of a protein and that the
benefit of pegylating a protein can be dependent upon
the site of modification. (The net average WBC area
under the curve after the single subcutaneous injection

35 (calculated according to CRC Standard Mathematical

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Tables, 26th Ed. (Beyer, W.H., Ed.) CRC Press Inc., Boca
Raton, FL 1981. p. 125) was similar for the Lys-35 and
N-terminal monopegylated species.)

5 e. stability studies

In addition, stability studies were performed
on the N-terminal and Lys-35 monopegylated species as
prepared above. (The Lys-41 material was not used as it
demonstrated no additional activity beyond unmodified

10 G-CSF) . These studies demonstrate that the N-terminally
pegylated G-CSF is unexpectedly more stable upon storage
than the other form of monopegylated G-CSF,
monopegylated lysine 35. Stability was assessed in
terms of breakdown of product, as visualized using

15 SEC-HPLC.

Methods: N-terminally pegylated G-CSF and lysine-35
monopegylated G-CSF were studied in two pH levels,
pH 4.0 and pH 6.0 at 4°C, each for up to 16 days.
Elevating the pH to 6.0 provides an environment for

20 accelerated stability assays. For the pH 6.0 samples,
N-terminal monopegylated G-CSF and Lysine 35
monopegylated G-CSF as prepared above were placed in a
buffer containing 20 mM sodium phosphate, 5 mM sodium
acetate, 2.5 % mannitol, 0.005 % Tween-80, pH 6.0 at a

25 final protein concentration of 0.25 mg/ml. One ml

aliquots were stored in 3 ml sterile injection vials.
Vials of each was stored at 4°C and 29°C for up to
16 days. Stability was assessed by SEC-HPLC tracings.
If the later measurements stayed the same (as

30 ascertained by visual inspection) as the initial (Time =
0) measurements, the sample was considered to be stable
for that length of time.

Results: Results are illustrated in FIGURES 6A-6C.
(a) Comparison at pH 6.0 at 4°C. FIGURE 6A shows the
35 4°C SEC-HPLC profiles for N-terminally monopegylated

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G-CSF at pH 6 over time and FIGURE 6B shows the 4°C
SEOHPLC profiles for lysine-35 monopegylated G-CSF at
pH 6 over time. One interpretation is that the Lys-35
material is breaking down to a material with a molecular
5 weight similar to that of unmodified G-CSF.

(b) Extended duration at pH 4.0 at 4°C. PH 4.0 and 4°C
provides something of a control illustrating relatively
stable conditions in that the N-terminal species shows
no degradation. For the Lys 35 species, the break down

10 of the material is still occurring, but at a much slower
rate.

(c) Comparison at pH 6.0 at 4°C. FIGURES 6C illustrates
the SEC-HPLC profiles for the monopegylated G-CSF' s
under these conditions, under extended time periods. As

15 can be seen, at pH 6.0 and 4°C, the lysine-35 material
exhibits no increase in depegylation at day 16 or day 35
beyond what was seen for day 6 (FIGURE 6B) . This
indicates that depegylation (instability) does not
change, under those conditions, beyond day 6.

EXAMPLE 2

This example demonstrates a method of
preparing a substantially homogenous population of

25 monopegylated G-CSF using reductive alkylation, and

characterization of this population. Recombinant G-CSF
as described in the above example was used. As can be
seen, not only do the present methods provide advantages
in terms of yield of N-terminally chemically modified

30 material, but also, the amine linkages of the present
reductive alkylation process produce substantially more
stable products as demonstrated by a large difference in
the degree of aggregation upon storage.

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A. Preparation of the Tnono-methoxypol yethvlene
glyrnl-KCSF rnnjngat.es with the site nf at t.anhment at
the M-terminal tt-amino residue.

To a cooled (4 °C) , stirred solution of rhG-CSF
5 (1 ml, 5 mg/ral as described in the Example above) in 100
mM sodium phosphate, pH 5, containing 20 mM NaCNBH3, was
added a 5- fold molar excess of methoxypolyethylene
glycol aldehyde (MPEG) (average molecular weight, 6 kDa) .
The stirring of the reaction mixture was continued at

10 the same temperature.

The extent of the protein modification during
the course of the reaction was monitored by SEC HPLC
using Bio-Sil SEC 250-5 column (BIO- RAD) eluted with
0.05 M NaH2PO4/0.05 M Na2HP04,0.15 M NaCl, 0.01 M NaN3,

15 pH 6.8 at 1 ml/min.

After 10 hours the SEC HPLC analysis indicated
that 92% of the protein has been converted to the
mono-MPEG-GCSF derivative. This can be seen in FIGURE 7,
which is a recording of the protein concentration (as

20 determined by absorbance at A280) and shows the peak
eluting at 8.72 minutes as monopegylated G-CSF, and a
minor peak of unreacted G-CSF eluting at 9.78 minutes.

As a comparison, FIGURE 8 shows the peaks
obtained when using N-hydroxysuccinimidyl ester of MPEG.

25 The molecular weight was also 6kDa. As can be seen, the
mixture obtained from this reaction was: t r i-MPEG-GCSF
conjugated (shoulder at approximately 7.25 minutes),
di-MPEG-GCSF conjugate (peak at 7.62 minutes),
mono-MPEG-GCSF conjugate (peak at 8.43 minutes) and

30 unreacted G-CSF (peak at 9.87 minutes) .

At this 10 hour time point, where 92% of the
protein had been converted to monopegylated material,
the pH of the reaction mixture was adjusted to pH 4 with
100 mM HC1 and the reaction mixture was diluted 5 times

35 with 1 mM HC1.

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The mono-MPEG-GCSF derivative was purified by
ion exchange chromatography using HiLoad 16/10 S
Sepharose HP column (Pharmacia) equilibrated with 20 mM
sodium acetate buffer, pH 4. The reaction mixture was
5 loaded on the column at a flow rate of 1 ml/min and the
unreacted MPEG aldehyde eluted with three column volumes
of the same buffer. Then a linear 400 minute gradient
from 0% to 45% 20 mM sodium acetate, pH 4, containing
1 M NaCl was used to the elute the protein-polymer
10 conjugate at 4°C.

Fractions containing the mono-MPEG-GCSF
derivative were pooled, concentrated and sterile
filtered.

Various mono-MPEG- GCSF conjugates obtained by
15 modifying rh-G-CSF with MPEG aldehydes of different
average molecular weight (12, 20 and 25 kDa) were
prepared in a similar manner.

■

b. Analysis of Monopegylated G-CSF

20 1. Molecular Weight

The molecular weight at the monopegylated
conjugates was determined by SDS-PAGE, gel filtration,
matrix assisted laser desorption mass spectrometry, and
equilibrium centrifugation. These results are presented

25 in Table 4, below.

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TABLE 4

Molecular Weiahts of N-

-tprminsnv Alkylated

Mono-MPEG-GCSF Conjugates

Conjugate

estimated

MW oel

filtration

MH maaa

apectomdtry

Hi ultra-
cantrif ugation

MPEG-
(6*Da)-
OCSF

24800

53024

24737

25548

MPEG-
(12kDa)-
GCSF

30800

124343

30703

29711

MPEG-
(20kOa) -
OCSF

38800

221876

38577

38196

»BG-
(25XDa>-
OCSF

43800

33326$

N/D

The structure of the prepared N-terminal
mono-MPEG-GCSF conjugates was confirmed using the
methods of N-terminal protein sequencing and peptide
mapping. Cyanogen bromide cleavage of the N-terminal
10 methionyl residue resulted in removal of the
polyethylene glycol.

2. Biological Activity
The in vitro biological activity of the
15 pegylated MPEG-GCSF con jugates 'was determined by

measuring the stimulated uptake of 3 H thymidine into

mouse bone marrow cells.

The in vivo biological activity was determined

by subcutaneous injection to hamsters MPEG-GCSF
20 conjugates or rhG-CSF (at lOOmg/kg) and measuring total
white blood cell count. Bioactivity as compared to
non-derivatized G-CSF was calculated as the area under

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the WBC/time curve after subtracting the vehicle control
curve. Relative bioactivity of the MPEG-GCSF derivatives
was expressed as the percentage bioactivity compared to
unmodified G-CSF.
5 This is illustrated in FIGURE 9, which is a

graph illustrating the total white blood cell response
to mono-N-terminal MPEG-GCSF conjugates prepared by
reductive alkylation of rhG-CF with MPEG aldehydes of
different molecular weights (6kDa, 12kDa, and 20kDa) .

10 As can be seen, all monopegylated molecules elicited a
response. The higher the molecular weight of the
polyethylene glycol moiety used, the higher the white
blood cell count achieved, except the 12kDa achieved a
slightly higher count than did the 20kDa version at

15 day 2.

3. Stability Studies

N-terminally pegylated G-CSF* s prepared by the
two different chemistries (amide vs. amine of the
20 reductive alkylation here) were compared for the degree
of aggregation. Unexpectedly, N-terminally pegylated
G-CSF using the amine chemistry was found to be
substantially more stable than N-terminally pegylated
G-CSF with an amide linkage (NHS chemistry as described

25 in Example 1) .

Methods: Both N-terminally pegylated G-CSF
samples were in 10 mM NaOac pH4.0 with 5% sorbitol, at a
concentration of Img protein/ml. The G-CSF 1 s were
pegylated with PEG 6000 for each. The amide-linked

30 conjugate was prepared as in Example 1, and the amine
linked conjugate was prepared as in Example 2. Six
samples of each were stored for eight weeks at 45°C. At
the end of eight weeks, the degree of aggregation was
determined using size exclusion chromatography and ion

35 exchange chromatography.

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Results: The results demonstrate that the
present reductive alkylation methodis advantageous over
aceylation because, surprisingly, it produces a material
with far fewer aggregates after 8 weeks at elevated
5 temperatures. The table below shows the percent of

non-aggregated material ("main peak" material) for both
materials using size exclusion chromatography (SEC) or
ion exchange (IE) :

10 TABLE 5

Sample: 8 wks, 45°C

% Main Peak
SEC/IE

Amine

82%/84%

Amide

37%/65%*

* This is relatively high because ion exchange does not
allow for full analysis of the aggregation.

15 EXAMPLE 3

This example demonstrates chemically modified
consensus interferon. More specifically, this example
demonstrates a method of preparing a substantially
20 homogenous population of monopegylated IFN-coni, and

characterization of this population.

It should be noted that while the present
example uses IFN-coni, any of the consensus interferons
as set forth above may be chemically modified. Such

25 chemical modification may be with any of the water

soluble polymers as listed above, although PEG is used
here. For pegylation, PEG 12000 is used here, although
any water soluble PEG species may be used (PEG 12000 was
selected for ease in handling and convenience) . Again,

30 a variety of means for chemical modification are

available (such as acetylation) but, for selective N-

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terminal chemical modification, such as N-terminal
pegylation, the present reductive alkylation method as
described in this example is preferred.

5 A. Preparation of Consensus Interferon

IFN-ctconi (here referred to as IFN-coni) as
described in Figure 2 of U.S. Patent No. 4, 695,623,
which is incorporated by reference in its entirety, was
used for the preparation of monopegylated consensus
10 interferon. The IFN-coni was produced by expression of
exogenous DNA in bacteria, and contained a methionyl
residue at the N-terminus.

B. PeffVlation Of Consensus Tntfirfprnn

15 To a cooled (4 °C) , stirred solution of IFN-

coni (3.45 mg/ml, containing 35.25% of the N-terminally
blocked form) in 100 mM sodium phosphate, pH 4.0,
containing 20 mM NaCNBH3 was added a 8-fold molar excess
of methoxypolyethylene glycol aldehyde (MPEG) (average

20 molecular weight 12 kDa) .

The extent of the protein modification during
the course of the reaction was monitored by reverse
phase HPLC using a polymer-based

poly (styrene/divinylbenzene) column, such as PLRP-S (PL
25 Separation Sciences Polymer Laboratories) .

After 10 hours the reverse phase HPLC analysis
indicated that 80% of the protein with unblocked a-amino

group at the N-terminus has been converted to the
MPEG-IFN-coni derivative.

30 At the 10 hour time point, the reaction

mixture was diluted 5 times with water and the
mono-MPEG-IFN-Coni derivative was purified by ion
exchange chromatography using HiLoad 16/10 S Sepharose
HP column (Pharmacia) equilibrated with 20 mM sodium

35 acetate buffer, pH 4.0. The reaction mixture was loaded

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on the column at a flow rate of 1 ml/min and the
unreacted MPEG aldehyde eluted with three column volumes
of the same buffer. Then a linear 420 minute gradient
from 0% to 75% of 20 mM sodium acetate, pH 4.0,
5 containing 1 M NaCl was used to the elute the protein-
polymer conjugate at 4°C.

Fractions containing the mono-MPEG- IFN-Coni

derivative were pooled, concentrated and sterile
filtered.

C. Analysis of Monopeovlated Consensus Interferon

l. Homogeneity

The homogeneity of the purified
mono-MPEG- IFN-Coni conjugates was determined by SDS-PAGE

15 using 10-20% or 4-20% precast gradient gels (Integrated
Separation Systems) . The gels showed a main band at MW
35 kDa.

To characterize the effective size
(hydrodynamic radius) of each mono-MPEG- I FN* con i species

20 a Superose 6 HR 10/30 (Pharmacia) gel filtration column
was used. Proteins were detected by UV absorbance at
280 nm. The BIO-RAD gel filtration standards served as
globular protein molecular weight markers.

The structure of the purified N-terminal

25 mono-MPEG- IFN-coni conjugates was confirmed using the

methods of N-terminal protein sequencing and peptide
mapping .

It is noted that this IFN-coni preparation
contained some N-terminally blocked material, and this

30 material was not pegylated. The material which was

pegylated, however, was monopegylated at the N-terminus.
Thus, in this type of situation, one may wish to use
other means to separate the blocked from the unblocked
material, such as ion exchange or size exclusion

35 chromatography .

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2. Biological Activity
The in vitro biological activity of the
mono-MPEG- IFN Coni conjugates was determined by
5 measuring their antiviral bioactivity. The in vitro

biological activity of the mono-MPEG- IFN-Coni conjugates
was determined by measuring their antiviral bioactivity
in human (HeLa) cells.

It was found that the mono-MPEG (12 kDa) -IFN-
10 Coni conjugate shows 20% in yjjtxa bioactivity (in U/mg

of protein) when compared to the unmodified species. As
noted above for pegylated G-CSF, the in vitro assays,

while useful to demonstrate biological activity, may
show a rather low level of activity for chemically
15 modified proteins because of characteristic sustained
release. The in vivo biological activity may be higher
than the in vitro biological activity.

D. Chemically modified consensus interferon with
20 thP N-terminallv blocked molecules removed

The present reductive alkylation was also
performed on the above IFN-coni which had the portion of

N-terminally blocked molecules pre-removed. Both PEG
12000 and PEG 20000 were used in the reductive
25 alkylation method as described above.

The molecular apparent molecular weights were

as follow:

Conjuaate

Apparent MW by
Gel Filtration

Apparent MW by
SDS-PAGE

monoMPEG (12kDa)
IFN- coni

104.0 kDa

35.6 kDa

monoMPEG (20kDa)
IFN-coni

175.1 kDa

55.4 kDa

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Analysis of the IFN-coni 20 kDa PEG conjugate
using FPLC ion exchange chromatography resulted in three
peaks :

MonoMPEG- IFN-coni: 66% of the total area

5 (eluting at 265.93 ml)

Protein aggregate and oligo MPEG-IFN-coni

conjugate: 24% of the total area (eluting at 238.42
ml ) ; and

Unreacted IFN-coni: 10% of the total area

10 (eluting at 328.77 ml).

The conditions were not further optimized. One
may further separate the monopegylated material using
chromatographic or other methods.

15 While the present invention has been described

in terms of preferred embodiments, it is understood that
variations and modifications will occur to those skilled
in the art. Therefore, it is intended that the appended
claims cover all such equivalent variations which come

20 within the scope of the invention as claimed.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

U) APPLICANT: AMGEN INC.

(ii) TITLE OF INVENTION: N-Terminally Chemically Modified Protein

Composition and Methods

(iii) NUMBER OF SEQUENCES: 2

<iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Amgen Inc.

(B) STREET: 1640 Dehavilland Drive

(D) STATE: California
<E) COUNTRY: USA

(F) ZIP : 91320

(v) COMPUTER READABLE FORM:

(A> MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

<C> OPERATING SYSTEM: PC-DOS /MS-DOS

(D) SOFTWARE: Patent In Release #1.0, Version #1.25

<vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:
(C> CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Pessin, Karol M.

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 531 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

<ii) MOLECULE TYPE: cDNA

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<xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGACTCCAT TAGGTCCTGC TTCTTCTCTG CCGCAAAGCT TTCTGCTGAA ATGTCTGGAA 60

CAGGTTCGTA AAATCCAGGG TGACGGTGCT GCACTGCAAG AAAAACTGTG CGCTACTTAC 120

AAACTGTGCC ATCCGGAAGA GCTGGTACTG CTGGGTCATT CTCTTGGGAT CCCGTGGGCT 180

CCGCTGTCTT CTTGTCCATC TCAAGCTCTT CAGCTGGCTG GTTGTCTGTC TCAACTGCAT 240

TCTGGTCTGT TCCTGTATCA GGGTCTTCTG CAAGCTCTGG AAGGTATCTC TCCGGAACTG 300

GGTCCGACTC TGGACACTC* GCAGCTAGAT GTAGCTGACT TTGCTACTAC TATTTGGCAA 360

CAGATGGAAG AGCTCGGTAT GGCACCAGCT CTGCAACCGA CTCAAGGTGC TATGCCGGCA 420

TTCGCTTCTG CATTCCAGCG TCGTGCAGGA GGTGTACTGG TTGCTTCTCA TCTGCAATCT 480

TTCCTGGAAG TATCTTACCG TGTTCTGCGT CATCTGGCTC AGCCGTAATA G 531
(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 175 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu
1 5 10 15

Lys Cys Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu

20 25 30

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
35 40 45

Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser
50 55 60

Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His
65 70 75 80

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie

85 90 95

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Ser Pro Glu Leu Gly Pro Thr Leu

100

Asp Phe Ala Thr Thr lie Trp Gin
115 120

Pro Ala Leu Gin Pro Thr Gin Gly
130 135

Phe Gin Arg Arg Ala Gly Gly Val
145 150

Phe Leu Glu Val Ser Tyr Arg Val

165

Asp Thr Leu Gin Leu Asp Val Ala
105 110

Gin Met Glu Glu Leu Gly Met Ala

125

Ala Met Pro Ala Phe Ala Ser Ala

140

Leu Val Ala Ser His Leu Gin Ser
155 160

Leu Arg His Leu Ala Gin Pro
170 175

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WHAT IS CLAIMED IS:

1. A substantially homogenous preparation of
N-terminally chemically modified G-CSF or analog
5 thereof, optionally in a pharmaceutical^ acceptable
diluent, carrier or adjuvant.

2 A preparation of claim 1 where said G-
CSF is chemically modified with a chemical selected from
10 the group consisting of dextran, poly(n-vinyl

pyurrolidone) , polyethylene glycols, propropylene glycol
homopolymers, prolypropylene oxide/ethylene oxide co-
polymers, polyoxyethylated polyols and polyvinyl
alcohols.

3. A preparation of claim 2 where said G-CSF
or analog thereof is chemically modified with
polyethylene glycol.

20 4 . A preparation of claim 3 said

polyethylene glycol has a molecular weight of between
about 2 kDa and 100 kDa.

5. A preparation of claim 4 wherein said
25 polyethylene glycol has a molecular weight of between
about 6 kDa and 25 kDa.

6. A preparation of claim 1 wherein said
preparation is comprised of at least 90% N-terminally

30 raonopegylated G-CSF or analog thereof and at most 10%
unpegylated G-CSF or analog thereof.

7. A preparation of claim 6 wherein said
preparation is comprised of at least 95% N-terminally

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monopegylated G-CSF or analog thereof and at most 5%
unpegylated G-CSF or analog thereof.

8. A preparation of claim 1 whererein said
5 G-CSF has the sequence identified in SEQ. ID No. 1.

9. A substantially homogenous preparation of
N-terminally monopegylated G-CSF, optionally in a
pharmaceutical ly acceptable diluent, carrier or

10 adjuvant, wherein: (a) said G-CSF has the amino acid

sequence identified in SEQ. ID No. 1; (b) said G-CSF is
monopegylated with a polyethylene glycol moiety having a
molecular weight of about 12 kDa.

15 10. A pharmaceutical composition comprising:

(a) a substantially homogenous preparation of
monopegylated G-CSF, said monopegylated G-CSF consisting
of a polyethylene glycol moiety having a molecular
weight of about 12 kDa connected to a G-CSF moiety

20 solely at the N-terminus thereof via an amine linkage;

(b) fewer than 5% non-pegylated G-CSF molecules; and (c)
a pharmaceutially acceptable diluent, adjuvant or
carrier.

25 11. A method of treating a hematopoietic

disorder comprising administering a therapeutically
effective dose of a preparation of any of claims 1-10.

12. A method for attaching a water soluble
30 polymer to a protein or analog thereof, wherein said
water soluble polymer has a single reactive aldehyde
group, said method comprising:

(a) reacting a protein moiety with a water
soluble polymer moiety under reducing alkylation
35 conditions, at a pH sufficiently acidic to selectively

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activate the a-amino group at the amino terminus of said

protein moiety so that said water soluble polymer
selectively attaches to said a-amino group; and

(b) obtaining the reaction product and

13. A method of claim 12 wherein said polymer
is pharmaceutical^ acceptable.

14. A method of claim 12 wherein said water
soluble polymer is selected from the group consisting of
dextran, poly(n-vinyl pyurrolidone) , polyethylene
glycols, propropylene glycol homopolymers,
15 prolypropylene oxide/ethylene oxide co-polymers r
polyoxyethylated polyols and polyvinyl alcohols.

15. A method of claim 14 wherein said polymer
is polyethylene glycol.

16. A method of claim 12 wherein said
reducing alkylation reaction involves the use of a
reducing agent selected from sodium borohydride, sodium
cyanoborohydride, dimethylamine borate, timethylamine

25 borate and pyridine borate.

17. A method for attaching a polyethylene
glycol molecule to a G-CSF molecule, wherein said
polyethylene glycol molecule has a single reactive

30 aldehyde group, said method comprising:

(a) reacting said G-CSF with said
polyethylene glycol molecule under reducing alkylation
conditions, at a pH sufficiently acidic to selectively
activate the a-amino group at the amino terminus of said

35 G-CSF; and

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(b) obtaining the pegylated G-CSF and

5 18. A method of claim 17 wherein said

polyethylene glycol molecule has a molecular weight of
about 6 kDa to about 25 kDa.

19. The pegylated G-CSF product produced by
10 the process of claim 17.

20. Chemically modified consensus interferon
comprised of a consensus interferon protein moiety
connected to at least one water soluble polymer moiety.

21. A chemically modified consensus
interferon of claim 20 wherein said consensus interferon
moiety is selected from the group consistiong of IFN-
coni, IFN-con2# and IFN-con3.

22. A chemically modified consensus
interferon of claim 21 wherein said water soluble
polymer is pharmaceutical^ acceptable.

25 23. A chemically modified consensus

interferon of claim 20 wherein said water soluble
polymer is selected from the group consisting of
dextran, poly(n-vinyl pyurrolidone) , polyethylene
glycols, propropylene glycol homopolymers,

30 prolypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols and polyvinyl alcohols.

24. A chemically modified consensus
interferon according to claim 23 wherein said water
35 soluble polymer moiety is polyethylene glycol.

WO 96/11953

PCT/US95/01729

- 53 -

25. A chemicaly modified consensus interferon
according to claim 20 wherein said water soluble polymer
moiety is connected to said consensus interferon moiety

5 directly without an additional linkage group,

26. A chemically modified consensus
interferon comprised of IFN-coni connected to at least

one polyethylene glycol moiety.

27. Pegylated consensus interferon.

28. A method for attaching a water soluble
polymer to consensus interferon, wherein said water

15 soluble polymer has a single reactive aldehyde group,
said method comprising:

(a) reacting a consensus interferon moiety
with a water soluble polymer moiety under reducing
alkylation conditions, at a pH sufficiently acidic to

20 selectively activate the a-amino group at the amino

terminus of said consensus interferon moiety; and

(b) obtaining the reaction product and

29. A method of claim 28 wherein said polymer
is pharmaceutical ly acceptable.

30. A method of claim 28 wherein said water
30 soluble polymer is selected from the group consisting of

dextran, poly(n-vinyl pyurrolidone) , polyethylene
glycols, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers,
polyoxyethylated polyols and polyvinyl alcohols.

WO 96/11953

PCIYUS95/01729

- 54 -

31. A method of claim 30 wherein said polymer
is polyethylene glycol.

32. A method of claim 28 wherein said

5 reducing alkylation reaction involves the use of a

reducing agent selected from sodium borohydride, sodium
cyanoborohydride, dimethylamine borate, timethylamine
borate and pyridine borate.

10 33. A method for attaching a polyethylene

glycol molecule to a consensus interferon molecule,
wherein said polyethylene glycol molecule has a single
reactive aldehyde group, said method comprising:

(a) reacting said consensus interferon with

15 said polyethylene glycol molecule under reducing

alkylation conditions, at a pH sufficiently acidic to
selectively activate the cc-amino group at the amino

terminus of said consensus interferon; and

(b) obtaining the pegylated consensus
20 interferon and

25 34. A method of claim 33 wherein said

polyethylene glycol molecule has a molecular weight of
about 2 kDa to about 100 kDa.

35. The pegylated consensus interferon
30 product produced by the process of claim 33.

36. A substantially homogenous preparation of
monopegylated consensus interferon.

WO 96/1 1953

PCI7US95/D1729

- 55 -

37. A preparation of claim 36 comprising
about 90% raonopegylated consensus interferon and about
10% unpegylated consensus interferon.

5 38. A pharmaceutical composition comprising:

(a) a substantially homogenous preparation of
monopegylated consensus interferon, said monopegylated
consensus interferon consisting of a polyethylene glycol
moiety connected to a consensus interferon moiety solely
10 at the N-terminus thereof via an amine linkage; (b)
fewer than 5% non-pegylated consensus interferon
molecules; and (c) a pharmaceutially acceptable diluent,
adjuvant or carrier.

WO 96/11953

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PCT/US95/01729

WO 96/11953

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PCT/US95/0172!)

Fig. 1 B

Lane No. 12 3 4 5 6
Lane No, Sample

MW Protein Standards

«r

rHuG-CSF Std

3.0

SCM-PEG-GCSF Reaction Mix

10.0

Species 1 (N-Term)

10.0

Species 2 (Lys-35)

10.0

Species 3 (Lys-41)

•

10.0

SUBSTITUTE SHEET (RULE 26)

BEST AVAILABLE COPY

WO 96/11953

3/15

PCT/US9S/01729

Fig. 2

SEC-HPLC Profiles of

(A) rHuG-CSF standard

(B) SCM-PEG-GCSF Reaction Mixture

(D) Species 2 (Lys-35 Derivative)

(E) Species 3 (Lys-41 Derivative)

O60 080 UK> TSo 1.40 TS)

x 10 minutes

SUBSTITUTE SHEET (RULE 26)

WO 96/11953

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P9|B0S

SUBSTITUTE SHEET (RULE H)

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SUBSTITUTE SHEET (RULE 26)

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pe|Bos

SUBSTITUTE SHEET (RULE 26)

W096/119S3

7/15

PCIYUS95/01729

mm^m

• HHP

LO-
CO

100
90
80
70
60

50
40
30
20
10
0

100

Fig. 4

NONE- native
GCSF

N-Term

Lys35
(unstable*)

Site of Modification

Lys41

* contains de-Pegylated rHuG-CSF, generated during storage.

SUBSTITUTE SHEET (RULE 26)

WO 96/11953

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(in/siiaogvOI.) 09M

SUBSTITUTE SHEET (RULE 26)

WO 96/11953

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(-jn/sABp x s||90 evOO (e6BJ9Av) OOV

SUBSTITUTE SHEET (RULE 26)

WO 96/1 1953

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10/15

Fig. 6A

N-term monopegylated G-CSF, pH 6

8.00-

6.00-

4.00-

2.00-

0.00 -

T=0 days

T=4 days

T=6 days

T=12 days

T=1 6 days

0.60 0.80

1.60

x10 minutes

SUBSTITUTE SHEET (RULE 26)

WO 96/11953

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Fig. 6B

Lys 35 monopegylated G-CSF, pH 6

8.00-

^ 6.00-
o

2 4.00 H

2.00-

0.00-

T=0 days

T=4 days

T=© days

T=12days

T=1 6 days

0.60

0.80

1.00

1.60

x10 1 minutes

SUBSTITUTE SHEET (RULE 26)

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Fig. 6C

Lys 35 monopegylated G-CSF, pH 7

SUBSTITUTE SHEET (RULE 26)

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Fig. 7

SUBSTITUTE SHEET (RUL€ 26)

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Fig. 8

SUBSTITUTE SHEET (RULE 26)

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Fig. 9

0 1 2 3 4 5

Days

SUBSTITUTE SHEET (RULE 26)

INTERNATIONAL SEARCH REPORT

tnitioud Application No

PCT/US 95/01729

A. CLASSIFICATION OF SUBJECT MATTER

IPC 6 C07K14/53 C07K14/555 C07K1/107 A61K47/48

Acconing to International Patent
B. FIELDS SEARCHED

Minimum documentation searched (dam ficaOon system followed by damftcafion symbols)

IPC 6 C07K A61K

Documentation searched other than minimum documentation to the extent that such document! are included in the fields

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

with indication, where appropriate, of the relevant passages

Relevant to claim No.

EP.A.O 098 110 (NIHON CHEMICAL RESEARCH
KABUSHIKI KAISHA) 11 January 1984

* example 3; page 4, 11nel3 to page 5,
line 13 *

W0.A.90 04606 (ROYAL FREE HOSPITAL SCHOOL
OF MEDICINE) 3 May 1990

* whole disclosure *

W0.A.89 05824 (GENETICS INSTITUTE) 29 June
1989

* example 9; pages 1-2 *

-/--

31-38

3-11,

15-19,

31-38

3-11.
15-19

Further documents are

in the continuation of box C.

Patent family members arc listed io annex

" Special categories of died doe wn c att :

'A" document defining the general state of the art which is not
considered to be of particular relevance

*E" earlier document but published on or after the interntnonil
filing date

*V document which may throw doubts on priority daunfs) or
which is deed to establish the publication date of another
dtation or other special reason (as specified)

*0* document referring to an oral dmsoswc, use, exhibition or

T* later document published after the international filing date
or priority date and not in conflict with the application but
dted to understand the principle or theory underlying the

"P* docurncnt published prior to the international filing date but
later than the priority date claimed

"X* document of particular relevance; the datmcd invention
cannot be considered novd or cannot be considered to
involve an inventive step when the document is taken alone

"Y* document of particular rdevance; the daimed invention
cannot be considered to involve an inventive step when the
document is comhinrd with one or more other such docu-
ments, wen combination being obvious to a person skilled
in the art.

"4" document member of the same patent family

Date of the actual completion of the international search

28 June 1995

Date of mailing of the

search report

08. It. 35

of the ISA

European Patent Office, P.B. 511 S Patenflaan 2
NL - 22X0 HV Rijrwi*
Td. ( + 31-70) 340-2040, Tx_ 31 651 cpo nl.
Fax (+ 31-70) 340.3016

Authorized officer

HERMANN R.

Form PCT/tSA/311 1

page 1 of 2

INTERNATIONAL SEARCH REPORT

CXCcoamudoci) DOCUMENTS CONSI DERED TO BE RELEVANT
Category * I Qtti<» of tWiimmL with where appropriate, of the relevant paatarci

FOCUS ON GROWTH FACTORS,
vol .3,

pages 4-10

FRANCIS, G.E. 'Protein modification and
fusion proteins 1
cited in the application

* whole disclosure *

B I OCON JUGATE CHEN.,
Vol.5,

pages 133 - 140

CHAMOW, S.M. ET AL. 'Modification of CD4
immunoadhesin with raonomethoxypoly-
(ethyleneglycol) aldehyde via reductive
alkylation 1

* table 1; figure 2; discussion *

L. STRYER "Biochemistry (2nd edition) 1 ,
FREEMAN & CO. , SAN FRANCISCO

* page 80, table 4-1 *

'Biochemica Katalog 1994, page 362 1 ,
BOEHRINGER MANNHEIM , MANNHEIM (GER)

*roa&onal Apctication No

PCT/US 95/01729

i Relevant to claim No.

3-11,
15-19,
31-38

17,33

Fore PCI7UA/21I «

*«t) (July IW)

page 2 of 2

INTERNATIONAL SEARCH REPORT

Information oo patent family mcmbcri

Patent document
cited in search report

Publication
date

croational Application No

PCT/US 95/01729

Patent family
membcr(s)

Publication
date

EP-A-0098110

11-01-84

JP-A-
JP-C-
JP-B-
JP-A-
US-A-

58225025
1784880
4063053

59059629
4609546

27-12-83
31-08-93
08-10-92
05-04-84
02-09-86

WO-A-9004606

03-05-90

EP-A-
JP-T-
US-A-

0439508
4501260
5349052

07-08-91
05-03-92
20-09-94

W0-A-8905824

29-06-89

US-A-
AU-B-
EP-A-

4904584
2911189
0355142

27- 02-90
19-07-89

28- 02-90

F«m PCT/ISA/Ui (fwunl tanily mom) l«)

INTERNATIONAL SEARCH REPORT

International application No.

PCT/ US 95/ 01729

Box I Observations where certain claims were found unsearchable (Continuation of Hem I of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) Tor the following reasons:
1. n Claims Nos-

because they relate to subject matter not required to be searched by this Authority, namely:

2. Claims No*.:

because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:

3. Q Claims Nos.:

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box II Observations where unity of invention it lacking (Continuation of item 2 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

- see additional sheet ISA/210

1. | 1 As all required additional search fees were timely paid by the applicant, this international search report covers aU

searchable claims.

2. 1 1 As all searchable claims could be searches without effort justifying an additional fee, this Authority did not invite payment

of any additional fee.

3. | I As only some of the required additional search fees were timely paid by the applicant, this international search report
covers only those claims for which fees were paid, specifically claims Nos.:

4. | X | No required additional search fees were timely paid by the applicant Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

3-10,15-19,31-38: 11 partially

| ] The additional search fees were accompanied by the applicant's protest
[ | No protest accompanied the payment of additional search fees.

Form PCT7ISA/210 (continuation of first sheet (1)) (July 1992)

international Application No. PCT/US95/ 01729

FURTHER INFORMATION CONTINUED FROM PCT/ISA/

- claims 3-10,15-19,31-38; 11 partially

Method for N-terra1nal PEGylatlon; monoPEGylated G-CSF or conINF, and claims
relating to said compounds

- claims 1,2,12-14,28-30; 11 partially

Method for N-term1nal modification (other than PEGylatlon); N-term1nally
modified G-CSF or conINF, and claims relating to said compounds

- claims 20-27; 11 partially

Unspedf ically PEGylated conINF, and claims relating to said compound

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